Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system.

Benjamin Garcia-Bloj,Rabab Rashwan,Ricardo L Mancera,Anabel Sorolla,Ciara Duffy,Andrea Leisewitz,Janice Plani-Lam,Agustin Sgro,Mahira Arooj,Theresa Swift-Scanlan,Alejandro H Corvalan,Shreyas Thiruvengadam,Colette Moses,Pilar Blancafort

doi:10.18632/oncotarget.11142

Abstract

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Highlights

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role in driving both cancer initiation and tumor evolution
We explored the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ deactivated’ Cas9 (dCas9) system in conjunction with protein-based artificial transcription factors (ATFs), Zinc Fingers (ZFs) proteins and Transcription Activator-Like Effectors (TALEs), to reactivate two dormant class II TSGs, and determined optimized combinatorial strategies able to functionally reactivate tumor suppression in cancer cell models
ZFs, TALEs and CRISPR/dCas9 were each C-terminally linked with the transcriptional activator VP64 (Figure 1A)

Summary

Introduction

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role in driving both cancer initiation and tumor evolution. In contrast with class I TSGs, which are frequently associated with loss of function alterations, class II TSGs are solely downregulated by epigenetic mechanisms, such as DNAand histone-posttranslational modifications, and are not altered at the genomic level [1]. The reversible nature of these epigenetic alterations offers unique opportunities for potential therapies. Gene reactivation of silenced TSGs remains a challenge, as it must overcome the threshold necessary to regain gene function and achieve cancer cell phenotypic reprogramming [2]. There is a demand for highly selective activation strategies that are able to upregulate the expression of otherwise dormant genes

Methods

Results

Conclusion